MICROBIAL BETA-GALACTOSIDASE: a SURVEY for NEUTRAL Ph OPTIMUM ENZYMES
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]. Milk Food Technol, Vol. 37, No. 4 (1974) 199 MICROBIAL BETA-GALACTOSIDASE: A SURVEY FOR NEUTRAL pH OPTIMUM ENZYMES L. C. BLANKENsHIP1 AND P. A. WELLS Dairy Foods Nutrition Laboratory, Nutrition Institute Agricultural Research Center, ARS, U. S. Departmant of Agriculture Beltsville, Maryland 20705 (Received for publication November 19, 1973) ABSTRACT MATERIALS AND METHODS Downloaded from http://meridian.allenpress.com/jfp/article-pdf/37/4/199/2399667/0022-2747-37_4_199.pdf by guest on 02 October 2021 Pure cultures of yeast, molds, and bacteria were screened Cultures and media for neutral pH optimum .B-galactosidases ( lactases) that would One hundred twenty-five ·identified yeasts, molds, and bac be suitable in dairy l;lroducts applications. Only 2 of 125 teria were obtained from the culture collection of the North identified and 10 of 250 unidentified cultures warranted fur em Regional Research Center, Peoria, Illinois. Cultures of ther study. These cultures produced high levels of· .a-galacto the following genera were included: Penicillium, Aspergillus, sidase with moderate galactose product inhibition. Character Absidia, Cunninghamella, Mucor, Rhizopus, CircineUa, Blake ization of the partially purified enzymes from unidentified cul slea, Chlamydamucor, Streptomyces, Actinomyces, Actinopyc hwes revealed that all required either Na+, K+ or Mg++ nidium, Debaryomyces, Kluyveromyces, Pichia, Schwanniomy c::ation activation, were inhibited by Cu + +, Mn + +, and ces, Bullera, Brettanomyces, Candida, Cryptococcus, Rhoda Fe+ + +, were most active around pH 6.8, and were unstable torula, Torulopsis, Escherichia, and Bacillus. Additionally, during storage (at either - 196 C or 4 C} . except in the 250 unidentified organisms were isolated by enrichment cul presence of 0.5 M ammonium sulfate. Most of the enzymes ture techniques from soil samples taken from locations used compared favorably in performance with a commercially avail for dairy waste disposal. able .a-galactosidase when tested in skim milk. Yeasts were maintained on yeast extract-malt extract ( YM ) Renewed interest in enzymatic hydrolysis of lactose agar slants (5) having the following composition in grams per liter: yeast extract, 3.0; malt extract, 3.0; peptone, 5.0; in milk and dairy products has been stimulated by glucose, 10.0; and agar, 25.0. Molds were maintained on recent studies demonstrating the prevalence of lactose Czapek Dox agar (5). Bacteria were maintained on either intolerance among certain groups of consumers; (3, trypticase-soy agar (Baltimore Biological Lab) or yeast ex 7, 11). Furthermore, the pollution problem posed by b·act-peptone-lactose agar ( YPL) having the following com disposal of enormous quantities of cheese whey con position ,in grams per liter: yeast extract, 2.0; peptone, 10.0; ammonium sulfate, 4.0; lactose, 10.0; salts solution, 20.0 ml taining large amounts of lactose has forced food scien (2); and agar, 15. Cultures were stored at 2 C after in tists to seek more meaningful uses for whey. In cubation. creased use of whey in foods and feed, however, will necessarily be limited by consumer lactose tolerance Enrichment cultures and will consequently require some degree of lactose Enrichment cultures were grown in either YPL broth or in YM broth with lactose substituted for glucose. Each 1-2 g hydrolysis. of soil sample was inoculated into 200 ml of broth contained The feasibility of enzymatically hydrolyzing lactose in a 1-liter flask. Samples were incubated at 28 C. Samples in milk and dairy products has been reported by were periodically removed, diluted, and spread on appropri several laboratories (8, 14, 15). These studies have ate agar plates. Isolated colonies were picked and purified been carried out largely with yeast enzymes. Micro by isolation from additional spread plates. Isolates were identified as yeast, mold, or bacterium by gram strains and bial ,8-galactosidase, E.C. 3.2.1.22, (lactase) would phase contrast microscopy of wet mounts. appear to have the greatest commercial potential pri marily because of ease of production. Although a Screening for .a-galactosidase production broad variety of microorganisms are known to pro Two hundred-milliliter broth cultures contained in 1-liter duce ,8-galactosidase, (1, 9, 10, 13, 16, 17 ), very few flasks that were incubated for 24 to 48 h on a gyratory shaker have properties that would make them suitable for at 28 C were used for cell production during screening. The total mass of yeasts and bacteria was harvested by centri commercial use. Consequently we initiated a screen fugation, that of molds by filtration. Cells and mycelia were ing program to search for new sources of neutral pH washed once in 35 ml of 0.05 M potassium phosphate buffer optimum ,8-galactosidases that would be suitable for pH 6.8, and resuspended in fresh buffer to a 12-ml volume; dairy product applications. extracts were prepared by using a Sooifier cell disruptor, Model W 185 (Heat Systems - Ultrasonics, Inc., Plainview, N. Y. ). Sonication was done in an ice-water bath at 90-95 1Present address: Southeastern Regional Research Center, ARS, watts power setting. Particulate matter was removed by cen USDA, Athens, Georgia 30604. trifugation, and extracts were stored in liquid nitrogen. 200 Enzyme assay levels of e'Q.ZYIIle, and both were inhibited by galactose P-Galoctosidase activity of crude extracts was detennined in excess of 53%. These organisms, Klyveromyces by incubating the following reaction mixtll're for SO min at 35 C in screw cap tubes: enzyme; phosphate buffer pH 6.8, lactis NRRL Y-1118 and Klyveromyces fragilis NRRL 0.07 M; lactose, 0.139 M; and water in a total volume of 3.0 Y-1109, are known ,a-galactosidase producers and the ml. Parallel reactions were conducted in which 0.139 M D enzyme from Y-1109 has been well studied in dairy galactose was included to estimate product inhibition. Re products applications (15). actions were stopped by boiling 5 min. The amount of glu The remaining cultures producing substantial quan cose liberated was then determined according to the method described by Jasewicz (6). o-Nitrophenol-P-n-galactopyrano tities of ,B~galactosidase were isolated from enrich side ( ONPG) ( Calbiochem) was used as substrate for puri ment cultures. Crude extract specific activities, gal fication, thermal stability, pH optimum, activator, and in actose inhibition, enzyme yields, and morphological hibitor experiments. A unit of enzyme was defined as that type of the 10 most active organisms are given in amount which produced one .umole of glucose or 0-nitro Table 1. Generally, yeast enzymes were more sus phenol per minute under the reaction conditions specified above. Specific activity was defined as the number of units ceptible to galactose inhibition than were bacterial Downloaded from http://meridian.allenpress.com/jfp/article-pdf/37/4/199/2399667/0022-2747-37_4_199.pdf by guest on 02 October 2021 per mg protein. Protein was determined by the Biuret method enzymes. Since no attempt was made to determine (4). optimal cultural conditions, it is possible that higher Enzyme purification yields might have been obtained with other media. Selected cultures were grown in 2-liter quantities, and Failure to find any promising mold enzymes was not extracts of cells were prepared by using a French pressure surprising since most mold ,8-galactosidases have acid cell operated at 16,000 psi. Partial purification was done by pH optima and would not have been active at the fractional ammonium sulfate precipitation followed by DEAE screening pH. sephadex chromatography using a NaCl gradient elution in Although microbial ,8-galactosidases are generally pH 7.0, 0.05 M phosphate buffer. of intracellular origin, the advantages of processing Enzyme characterization an extracellular enzyme dictated screening of spent Thermal stability of partially purified enzymes was de broths for activity. No ,a-galactosidase activity was termined by heating microgram quantities of enzyme in 1.0 ml of water in screw cap tubes at the desired temperature for detected in any of the spent broths. 10 min. Samples were immediately cooled in an ice bath Microscopic examination of unidentified selected and the remainder of the reaction ingredients were added. cultures showed that all the bacteria were gram Reaction mixtures were then brought to 35 C, ONPG was negative rods. Yeasts were observed to be ellipsoidal added, and hydrolysis rates were determined from kinetic budding types. traces by using a recording Beckman DB spectrophotometer at 420 nm. Enzyme purification A variety of mono- and divalent cations were tested for Ammonium sulfate fractionation of selected culture activator or inhibitor effects on the selected, partially puri crude extracts generally resulted in precipitation of fied enzymes. These tests were conducted in tris-hydroxy methylamino methane chloride buffer pH 7.0 to avoid phos phate precipitates. TABLE 1. /1-GALACTOSIDASE PRODUCTION BY ENRICHMENT CULTURE ISOLATES Product inhibition studies with partially putified enzymes Unit!!/ were performed in which glucose or galactose was included Morpbologlcal Specific % Inhibition 200 ml in the standard reaction mixture at a final concentration of Culture type activity• by n-galactose culture 0.139 M. This corresponded to their concentration if total MTA-7 Bacterium 1.68 24.2 492 lactose hydrolysis had occurred in milk. MTB-28 Bacterium .476 29.3 116 pH optimum experiments were done in 0.05 M phosphate MTA-1 Bacterium 1.54 31.0 473 buffer over the range of 6 to 8. Results were normalized 105b Bacterium 3.40 36.6 1027 for differences in nitrophenol extinction coefficients at the Bel-17 Bacterium 1.91 13.2 600 various pH levels tested by reference to standard curves. Bel-15 Bacterium 1.22 29.1 411 MTB-18 Bacterium 1.38 38.0 224 REsULTS AND DISCUSSIOX 227 Yeast 2.14 51.6 534 217 Yeast 3.00 52.3 864 Screening 204 Yeast 3.71 49.5 682 The screening procedure used in this study was °Crude extracts.